Method for stabilizing duocarmycin derivatives

ABSTRACT

Stabilized duocarmycin derivatives represented by formula (I): ##STR1## wherein R is lower alkyl, allyl or benzyl, and X is Cl or Br, are prepared by adding at least a compound selected from the group consisting of a saccharide, an electrolyte, a water-soluble polymer, a polyhydric alcohol and a surfactant to a solution containing the duocarmycin derivatives. Also provided are freeze-dried pharmaceutical preparations containing the stabilized duocarmycin derivatives.

This application is a 371 of PCT/JP/95/00962 filed May 19, 1995.

TECHNICAL FIELD

The present invention relates to a method for the stabilization ofduocarmycin derivatives and to preparations containing such stabilizedderivatives.

BACKGROUND ART

Duocarmycin derivatives such as Compound A represented by the formulashown below are known to have anti-tumor activity (see U.S. Pat. No.5,070,092). In addition, Compound A is known to be stabilized in itshydrobromide form (see European Patent No. A° 537575). ##STR2##

However, duocarmycin derivatives including Compound A in itshydrobromide form easily decompose in an aqueous solution. Thus, theinstability of these compounds is a major problem in medicationsintended for long-term storage. Accordingly, a need exists for stablepharmaceutical preparations containing duocarmycin derivatives, whichcan be stored for a long period of time.

DISCLOSURE OF THE INVENTION

The present invention provides a method for stabilizing duocarmycinderivatives represented by general formula (I): ##STR3## wherein Rrepresents a lower alkyl group, an allyl group or a benzyl group, and Xrepresents a chlorine atom or a bromine atom, which method ischaracterized in that at least one member selected from the groupconsisting of a saccharide, an electrolyte, a water-soluble polymer, apolyhydric alcohol and a surfactant is present in a solution containingsaid duocarmycin derivatives. Preferably, in the method for thestabilization, a solution containing said duocarmycin derivativerepresented by formula (I) and a saccharide is freeze-dried. Morepreferably, a solution further containing at least one member selectedfrom the group consisting of an electrolyte, a water-soluble polymer, apolyhydric alcohol and a surfactant is freeze-dried. The presentinvention provides freeze-dried pharmaceutical preparations containingduocarmycin derivatives, which have been stabilized by theabove-mentioned method.

In the definition of formula (I), the lower alkyl group means a straightchain or branched alkyl group having 1 to 6 carbon atoms, for example, amethyl group, an ethyl group, a propyl group, an isopropyl group, abutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, anda hexyl group.

Duocarmycin derivatives represented by formula (I) can be produced bythe method described in U.S. Pat. No. 5,070,092.

Specific examples of duocarmycin derivatives for use in the presentinvention are shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________     ##STR4##                                                                     Compound No.                                                                          X  IR (KBr), ν (cm.sup.-1)                                         __________________________________________________________________________    1 (Compound A)                                                                        Br 3475, 3232, 2944, 1698, 1491, 1410, 1313, 1217, 1110               1 (Compound A)*                                                                       Br 1717, 1692, 1608, 1525, 1490, 1409, 1310, 1218, 1167, 1108         2       Cl 2940, 1698, 1637, 1491, 1410, 1314, 1218, 1154,                    __________________________________________________________________________               1109                                                                *Hydrobromide monohydrate                                                

In accordance with the present invention, a duocarmycin derivativerepresented by formula (I) and at least one member selected from thegroup consisting of a saccharide, an electrolyte, a water-solublepolymer, a polyhydric alcohol and a surfactant are dissolved in asolution, preferably an acidic solution, more preferably an acidicsolution that has been adjusted to pH 5 or less. The resulting solutionis filtered through a membrane filter under germ-free conditions andfreeze-dried.

As the saccharide, lactose, sucrose, raffinose, dextran, etc. may beused. Preferred is lactose. The concentration of the saccharide in thesolution is from 0.005 to 1000 mg/ml, preferably from 1 to 500 mg/ml.Any electrolyte which is pharmaceutically acceptable and which canincrease the ionic strength of the solution can be employed. Forinstance, inorganic acids such as hydrochloric acid, hydrobromic acid,hydroiodic acid, sulfuric acid, carbonic acid, silicic acid, phosphoricacid, and boric acid; organic acids such as citric acid, and aceticacid; and their sodium salts, potassium salts, etc. may be used. Theconcentration of the electrolyte in the solution is from 0.001 to 500mg/ml, preferably from 0.01 to 100 mg/ml. As the water-soluble polymer,polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol,carboxyvinyl polymer, etc. may be used at a concentration of 1 to 1000mg/ml, preferably 10 to 500 mg/ml. As the polyhydric alcohol, glycerin,propylene glycol, etc. may be used at a concentration of 10 to 1000mg/ml, preferably 100 to 500 mg/ml. As the surfactant, polyoxyethylenesorbitan fatty acid ester (Tween), polyoxyethylene-hydrogenated castoroil, etc. may be used at a concentration of 0.01 to 5000 mg/ml,preferably 0.1 to 500 mg/ml. The concentration of the duocarmycinderivative in the solution to be freeze-dried is from 0.001 to 1000mg/ml, preferably from 0.1 to 10 mg/ml. Freeze-drying of the solution iscarried out under appropriate conditions. For example, the solution isfirst frozen at -50° C. for 5 hours (pre-freezing), dried at -30° C. andat 0.05 mbar for 30 hours and then at 0° C. and at 0.05 mbar for 15hours (primary drying). The material then further dried at 25° C. and at0.05 mbar for 15 hours (secondary drying).

Typically, the solution is freeze-dried in a vial, and afterfreeze-drying, the vial containing the freeze-dried preparation issealed with a rubber stopper and an aluminium cap, whereby afreeze-dried pharmaceutical preparation containing a duocarmycinderivative is obtained.

When clinically using duocarmycin freeze-dried preparations, thefreeze-dried pharmaceutical preparation is dissolved in a solutioncontaining as a stabilizer at least one member selected from the groupconsisting of an electrolyte, a water-soluble polymer, a polyhydricalcohol and a surfactant.

The pharmaceutical preparation of the present invention may contain asmay be appropriate an antioxidant, an antiseptic, a buffer, a soothingagent, a solubilizer, an isotonicity agent, a preservative, astabilizer, a vehicle, a binder, a disintegrator, a wetting agent, alubricant, a coloring agent, an aromatic, a flavoring agent, a coating,a suspending agent, an emulsifier, a plasticizer, a surfactant, and thelike which are pharmaceutically acceptable. For instance, thepreparation may additionally be formulated to contain an antioxidantsuch as ascorbic acid, vitamin E, butylhydroxytoluene andbenzylhydroxytoluene; an antiseptic such as parabens and chlorobutanol;a buffer such as phosphoric acid and citric acid; a soothing agent suchas benzyl alcohol and lidocaine; a vehicle such as crystallinecellulose, hydroxypropyl starch, starch and corn starch; a binder suchas pullulan, polyvinyl alcohol and hydroxypropyl cellulose; adisintegrator such as carboxymethyl cellulose and croscarmellose sodium;a lubricant such as magnesium stearate, talc and hydrogenated oil, etc.

The pharmaceutical preparation of the present invention may be made notonly as injectable preparations, but also as oral preparations such astablets, capsules and granules, as well as for rectal administration inthe form of suppositories, etc.

The dose and administration schedule of the pharmaceutical preparationof the present invention for use as an anti-tumor agent will varydepending on various factors such as the patient's age, weight andphysical condition. For instance, when the preparation is used as ananti-tumor injection, it is suitable to administer the active compoundin an amount of 0.0001 to 10 mg/kg. Administration may be made once aday (single administration or consecutive administration) orintermittently once to three times a week or once every three weeks.

Certain specific embodiments of the invention are illustrated by thefollowing representative examples and test examples.

Best Mode for Carrying Out the Invention Example 1

In this example, 50 mg of citric acid, 100 mg of Compound A hydrobromideand 5000 mg of lactose were dissolved in distilled water to make a totalvolume of 200 ml. The resulting solution was put into 15-ml glass vials,in 2 ml portions, and freeze-dried under reduced pressure. Afterfreeze-drying, the pressure was restored to normal pressure in anitrogen stream, and each vial was sealed with a rubber stopper and analuminium cap to yield a freeze-dried preparation containing Compound A.

Example 2

In this example, 50 mg of citric acid, 100 mg of Compound Ahydrobromide, 5000 mg of lactose and 100 mg of sodium bromide weredissolved in distilled water to make a total volume of 200 ml. Theresulting solution was put into 15-ml glass vials, in 2 ml portions, andfreeze-dried under reduced pressure. After freeze-drying, the pressurewas restored to normal pressure in a nitrogen stream, and each vial waspealed with a rubber stopper and an aluminium cap to yield afreeze-dried preparation containing Compound A.

Example 3

In this example, 50 mg of citric acid, 100 mg of Compound Ahydrobromide, 5000 mg of lactose, 100 mg of sodium bromide and 500 mg ofpolyoxyethylene sorbitan monooleate (Tween 80) were dissolved indistilled water to make a total volume of 200 ml. The resulting solutionwas put into 15-ml glass vials, in 2 ml portions, and freeze-dried underreduced pressure. After freeze-drying, the pressure was restored tonormal pressure in a nitrogen stream, and each vial was sealed with arubber stopper and an aluminium cap to yield a freeze-dried preparationcontaining Compound A.

Example 4

In this example, 50 mg of citric acid and 100 mg of Compound Ahydrobromide were dissolved in distilled water to make a total volume of200 ml. The resulting solution was put into 15-ml glass vials, in 2 mlportions, and freeze-dried under reduced pressure. After freeze-drying,the pressure was restored to normal pressure in a nitrogen stream, andeach vial was sealed with a rubber stopper and an aluminium cap to yielda freeze-dried preparation containing Compound A.

Test Example 1

Compound A hydrobromide (1 mg) was dissolved in distilled water to makea total volume of 10 ml (control solution). Separately, 0.5 mg of citricacid and 1 mg of Compound A hydrobromide were dissolved in distilledwater together with 1 mg of sodium bromide (Test Solution 1), or 1 mg ofsodium bromide and 5 mg of Tween 80 (Test Solution 2) to make a totalvolume of 10 ml. All solutions tested had a pH adjusted to 3.6. Thecontrol solution and the test solutions were respectively put into 15-mlglass vials, and stored in a thermostat at 25° C. Each solution wassampled in an amount of 1 ml at predetermined intervals. The content ofCompound A remaining in each of the test sample solutions was analyzedby high performance liquid chromatography and the results are shown inTable 2.

Conditions for High Performance Liquid Chromatography

Column: Inertsil ODS-2, 6.0φ×250 mm

Mobile Phase: 0.05M phosphate buffer (pH 5.9)/acetonitrile (48 parts byvolume/52 parts by volume)

Flow Rate: 1.0 ml/min

Detection Wavelength: 330 nm

                  TABLE 2                                                         ______________________________________                                        Stability of Compound A in Aqueous Solution (at 25° C.)                          Content of Compound A (%)                                                       0 hour                                                            Test Solution                                                                             (start)     2 hours  4 hours                                      ______________________________________                                        Control Solution                                                                          100.0       95.5     91.9                                         Test Solution 1                                                                           100.0       95.7     93.2                                         Test Solution 2                                                                           100.0       98.6     97.9                                         ______________________________________                                    

As is seen from the results of the control solution in Table 2, CompoundA decomposes in an aqueous solution with the passage of time. Thestability of Compound A in an aqueous solution was improved by addingthereto sodium bromide alone (Test Solution 1) or with sodium bromideand Tween 80 (Test Solution 2). From these results, it is evident thatthe addition of sodium bromide or sodium bromide and Tween 80 to anaqueous solution of Compound A improves the stability of Compound A insolution during the preparation process of a freeze-dried preparationand during the period between the reconstitution of the freeze-driedpreparation and the administration of the reconstituted preparation to apatient. Thus, the probability of introducing a decomposition product ofCompound A to the patient is reduced.

Test Example 2

Citric acid (5 mg) and 1 mg of Compound A hydrobromide were dissolved indistilled water together with 1 g of propylene glycol (Test Solution 3)or 1 g of polyethylene glycol 400 (Test Solution 4) to make a totalvolume of 10 ml. Both solutions had a pH adjusted to 3.0. The testsolutions were respectively put into 15-ml glass vials, and stored in athermostat at 40° C. Each solution was sampled in an amount of 1 ml atpredetermined intervals. The content of Compound A remaining in each ofthe test sample solutions was analyzed by high performance liquidchromatography and the results are shown in Table 3.

Conditions for High Performance Liquid Chromatography

Column: Inertsil ODS-2, 6.0φ×250 mm

Mobile Phase: 0.05M phosphate buffer (pH 5.9)/acetonitrile (48 parts byvolume/52 parts by volume)

Flow Rate: 1.0 ml/min

Detection Wavelength: 330 nm

                  TABLE 3                                                         ______________________________________                                        Stability of Compound A in Aqueous Solution (at 40° C.)                         Content of Compound A (%)                                                       0 hour                                                             Test Solution                                                                            (start)      3 hours  6 hours                                      ______________________________________                                        Test Solution 3                                                                          100.0        68.4     46.8                                         Test Solution 4                                                                          100.0        84.9     70.8                                         ______________________________________                                    

Test Example 3

The freeze-dried preparations obtained in Examples 1 to 4 were stored ina thermostat at 60° C. for 30 days. The content of Compound A remainingin each of these preparations was analyzed by high performance liquid.10 Chromatography. The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Storage Stability of Compound A (at 60° C., 30 days)                   Example No. Content of Compound A (%)                                         ______________________________________                                        Example 1   100.0                                                             Example 2   100.4                                                             Example 3   99.3                                                              Example 4   97.6                                                              ______________________________________                                    

As is apparent from the results given in Table 4, the stability of thefreeze-dried preparations containing Compound A was improved by theaddition of the saccharide.

Industrial Applicability

The present invention provides a method for the stabilization ofduocarmycin derivatives and preparations containing them.

We claim:
 1. A method for stabilizing a compound, comprising the stepsof;selecting a compound represented by formula (I): ##STR5## wherein Rrepresents a straight chain or branched alkyl group having 1 to 6 carbonatoms, an allyl group of a benzyl group, and x represents a chlorineatom or a bromine atom; and preparing a solution of said compound and atleast one material selected from the group consisting of a saccharide,an electrolyte, a water-soluble polymer, a polyhydric alcohol and asurfactant.
 2. The method according to claim 1, wherein the solutioncontains said saccharide, and said method further comprises a step offreeze-drying said solution containing said compound and saidsaccharide.
 3. The method according to claim 2, wherein prior tofreeze-drying the solution also contains at least one additionalmaterial selected from the group consisting of an electrolyte, awater-soluble polymer, a polyhydric alcohol and a surfactant.
 4. Apharmaceutical composition which comprises a compound represented byformula (I): ##STR6## which is obtained by freeze-drying an acidicsolution containing the compound and at least one material selected fromthe group consisting of a saccharide, an electrolyte, a water-solublepolymer, a polyhydric alcohol and a surfactant.
 5. The freeze-driedpharmaceutical composition according to claim 4, wherein the solutioncontains the compound and said saccharide prior to freeze-drying.
 6. Thefreeze-dried pharmaceutical composition according to claim 5, whereinprior to freeze-drying the solution also contains at least one materialselected from the group consisting of an electrolyte, a water-solublepolymer, a polyhydric alcohol and a surfactant.